Selection of Shigella flexneri candidate virulence genes specifically induced in bacteria resident in host cell cytoplasm
| Autor: | Maria Chiara Pardini, Cecilia Bartoleschi, Carlo Pazzani, Maria Lina Bernardini, Claudia Scaringi, Maria Celeste Martino |
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| Rok vydání: | 2002 |
| Předmět: |
Cytoplasm
Operon Sequence analysis Immunology Chloramphenicol Resistance Virulence Viral Plaque Assay Microbiology Shigella flexneri Mice Plasmid Genes Reporter Virology Animals Humans Promoter Regions Genetic Gene Cells Cultured biology Gene Transfer Techniques Galactosidase activity biology.organism_classification Molecular biology Genes Bacterial Host cell cytoplasm HeLa Cells Plasmids |
| Zdroj: | Cellular Microbiology. 4:613-616 |
| ISSN: | 1462-5822 1462-5814 |
| DOI: | 10.1046/j.1462-5822.2002.00216.x |
| Popis: | Summary We describe an in vivo expression technology (IVET)- like approach, which uses antibiotic resistance for selection, to identify Shigella flexneri genes specifi- cally activated in bacteria resident in host cell cyto- plasm. This procedure required construction of a promoter-trap vector containing a synthetic operon between the promoterless chloramphenicol acetyl transferase ( cat ) and lacZ genes and construction of a library of plasmids carrying transcriptional fusions between S. flexneri genomic fragments and the cat-lacZ operon. Clones exhibiting low levels ( < 10 µ µ µ g ml − 1 ) of chloramphenicol (Cm) resistance on laboratory media were analysed for their ability to induce a cytophatic effect - plaque - on a cell mono- layer, in the presence of Cm. These clones were assumed to carry a plasmid in which the cloned frag- ment acted as a promoter/gene which is poorly expressed under laboratory conditions. Therefore, only strains harbouring fusion-plasmids in which the cloned promoter was specifically activated within host cytoplasm could survive within the cell mono- layer in the presence of Cm and give a positive result in the plaque assay. Pai ( p laque a ssay i nduced) clones, selected following this procedure, were anal- ysed for intracellular (i) β -galactosidase activity, (ii) proliferation in the presence of Cm, and (iii) Cm resis- tance. Sequence analysis of Pai plasmids revealed genes encoding proteins of three functional classes: external layer recycling, adaptation to microaerophilic environment and gene regulation. Sequences encod- ing unknown functions were also trapped and selected by this new IVET-based protocol. |
| Databáze: | OpenAIRE |
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